The metal ion effects of the activation, regulation, catalysis and inhibition are being investigated with three enzymes. The enzymes of interest are phosphoenolpyruvate carboxykinase (PEPCK) which plays a key role in gluconeogenesis, pyruvate kinase (PK) which plays a key role in glycolysis, and enolase. The PEPCK has a mercaptan requirement for maximum catalytic efficiency and its role in cation binding and catalysis will be investigated. The complex role of the cations in PEP and in nucleotide stereospecificity and binding will be investigated by kinetic and NMR studies. Comparative studies of PEPCK from several sources will be investigated using stereospecific substrate analogs. The cation binding sites of enolase will be studied by selective modification studies and by difference NMR spectroscopy. Details of catalytic interconversion and the substrate binding sites will be studied by 31P NMR. To elucidate the mechanism of allosteric activation of pyruvate kinase, binding isotherms of the ligands and the cations will be investigated by rapid flow and by EPR studies. The results of these studies will clarify several of the processes of enzyme activation and inhibition and will allow the understanding of metabolic regulation.